The overall goal of this competitive renewal continues to be to understand the mechanisms involved in maintaining the survival of breast carcinoma cells in hypoxia. The work funded by this grant has pioneered studies on the central role of autocrine VEGF in the survival of breast carcinoma cells and it revealed a key role for the VEGF receptor Neuropilin-1 (NP-1) in this survival. Significant progress was made in other areas as well, but this renewal focuses specifically on the hypothesis that the progression to invasive and metastatic breast carcinoma includes the elaboration of VEGFies signaling in tumor cells. Aim 1 will investigate the contribution of NP-1 to tumor formation and progression using conditional NP-1 knock out mice and the MMTV-MT model of mammary cancer. Also, the importance of NP-1 to the ability of human breast carcinoma cells to form xenografts and progress to metastasis will be assessed using shRNA. Aim 2 will address the novel hypothesis that a specific plexin, plexin A1, contributes to breast carcinoma survival by functioning as a co-receptor for NP-1 to mediate VEGF16s signaling. The hypothesis that NP-1/plexin A1 signaling functions to evade apoptosis by inhibiting GSK-3|3 and activating NFicB will also be pursued in Aim 2. Aim 3 will investigate the relationship between VEGFi65/NP-1 signaling and the dissolution of epithelial architecture that is associated with the genesis of invasive and metastatic cancers. Specifically, the hypothesis that normal mammary epithelial cells and highly differentiated carcinoma cells are independent of VEGF16s/NP-1 survival signaling but that these cells become dependent on VEGF-|65/NP-1 signaling during the epithelial to mesenchymal transition. In addition, the ability of hypoxia to stimulate an EMT by activating Akt, inhibiting GSK-Sp and inducing the expression of Snail, a transcription factor that represses E-cadherin will be studied. The contribution of VEGF to vascular and lymph angiogenesis dominated and, in fact, continues to dominate the study of this growth factor in breast and other cancers. Undoubtedly, this function of VEGF is essential. An important implication of our work, however, is that it challenges the notion that the function of VEGF in cancer is limited to angiogenesis and that therapeutic approaches based on the inhibition of VEGF function target only angiogenesis. In fact, therapy aimed at targeting either VEGF or VEGF receptors is showing considerable progress in clinical trials with late-stage cancer patients and the possibility that such therapy is targeting VEGF signaling in tumor cells is likely. For this reason, a rigorous understanding of the mechanisms used in this signaling and its relevance to breast cancer is essential.